Immunological method for detecting antibodies directed against tissue transglutaminase (tTG), use of tTG in diagnosis and therapy control, and an oral pharmaceutical agent containing tTG

ABSTRACT

The invention relates to a method for detecting antibodies from body fluids by means of an immune reaction with tissue transglutaminase (tTG), with tTG-containing compounds, the antigenic structures, immunoreactive sequences or analogues thereof. The method may be used in the diagnosis and therapy control of diseases associated with an immune reaction against tTG, tTG-containing compounds, the antigenic structures, immunoreactive sequences or analogues thereof. Therefore, the invention is also directed to the use of tTG and the above-mentioned substances in diagnosis and therapy control, preferably in the diagnosis and therapy control of chronically inflammatory diseases or autoimmune diseases, and more preferably, in the diagnosis and therapy control of sprue or coeliac disease.

[0001] The invention relates to a method for detecting antibodies frombody fluids by means of an immune reaction with tissue transglutaminase(tTG), the antigenic structures, immunoreactive sequences or analoguesthereof, and with tTG-containing compounds, the antigenic structures,immunoreactive sequences or analogues thereof. The method may be used inthe diagnosis and therapy control of diseases associated with an immunereaction against tTG, tTG-containing compounds, and antigenicstructures, immunoreactive sequences or analogues thereof. Therefore,the invention is also directed to the use of tTG and the above-mentionedsubstances in diagnosis and therapy control, preferably in the diagnosisand therapy control of chronically inflammatory diseases or autoimmunediseases, and more preferably in the diagnosis and therapy control ofsprue or coeliac disease. The invention is also directed to an oralpharmaceutical agent which includes tTG, tTG-containing compounds, theantigenic structures, immunoreactive sequences or analogues thereof asactive ingredients and may be employed in the treatment of diseasesaccompanied by an immune reaction against these substances, because oraladministration of the above-mentioned compounds results in an immunetolerance.

[0002] The present invention is based on the discovery that tissuetransglutaminase (tTG, EC 2.3.2.13) is the autoantigen of sprue orcoeliac disease.

[0003] On the basis of the above finding, the immunological method ofthe invention for detecting antibodies against tTG and tTG-containingcompounds has been developed.

[0004] Coeliac disease is a disease of the small intestine mucosa, thefirst manifestation predominantly occurring during the late infant andtoddler ages. If the corresponding clinical picture does not occurbefore the adult age, it is termed non-tropical sprue. Thus, both ofthese terms describe the same disease. Sprue is accompanied by aninflammatory change of the mucosa and a general malabsorption as aresult thereof. In most of the cases, there is a morphological andclinical response to a treatment using a diet free of gluten.

[0005] Well-known as pathogenic factors are glutens from wheat, barley,rye and, to some extent, oats, while those from plant types having alower degree of phylogenetic relation, such as corn, rice and soy arenon-pathogenic. Amongst said glutens, the role of the pathogenic agentis ascribed to the alcohol-soluble prolamins, specifically α-gliadin.

[0006] For this reason, sprue preferentially occurs in countries wherewheat is used as major source of food (Europe, U.S.A., Australia) andhas an incidence rate of 0.14/1,000 newborns in Denmark, 0.7/1,000 inSpain, 1/1,000 in Italy, 0.45/1000 in Germany, and 2.42/1,000 in Sweden,for example.

[0007] However, more recent investigations demonstrate that asubclinical pattern, i.e., a morphological change of the mucosa withoutmassive symptoms is more widespread than believed so far. Thus, a studycarried out in Italy in 1994 revealed an incidence of 3.28/1,000 amongschool children. The risk of latent sprue in the next of kin of spruepatients ranges up to 50%.

[0008] The predominantly latent sprue is frequently accompanied by apolymorphic dermatosis, i.e., Dermatitis herpetiformis, wherecharacteristic subepidermal small blisters with granular IgA deposits inthe dermal papillae tips can be observed. Biopsies of the smallintestine show an irregular, more or less severely damaged mucosa.

[0009] Another well-established association can be observed betweensprue and insulin-dependent Diabetes mellitus, thyroid gland diseases,and a selective IgA deficiency.

[0010] In addition to numerous concomitant clinical symptoms of sprue,such as anemia which, among other things, has been ascribed to a vitaminB₁₂ malabsorption, and a vitamin K deficiency representing the reasonfor an increased hemorrhage tendency, the massively increased risk ofgastrointestinal malignant tumors plays a special role. Up to 15% of thesprue patients, mostly at an age of more than 50 years, developneoplastic diseases, about 50% of which involving intestinal T celllymphomas and another 25% involving esophagus, oropharyngeal and smallintestine tumors.

[0011] The therapy of sprue comprises strict observance of a lifelonggluten-free diet, where not only gluten-containing products made ofwheat but also those made of rye, barley and oats must be excluded. Asfor the patients, this represents a grave restriction in both eatinghabits and social interactions.

[0012] If diagnosis and therapy of sprue are effected in time, there isa good prognosis. However, complications once having occurred arefrequently not completely reversible. Conversely, if the disease remainsunrecognized and untreated, severe symptoms may arise as a result ofmalabsorption. Ultimately, there is an increased risk of developingintestinal lymphomas and other gastrointestinal neoplasias.

[0013] At present, biopsy of the small intestine represents the toplevel standard in the diagnosis of sprue and the follow-up undergluten-free diet, but also non-invasive diagnostic methods based onimmunological markers become more and more important. Because IgA andIgG class antibodies are present in the serums of sprue patients, which,on the one hand, are directed against gliadin and, on the other hand,against an autoantigen of the endomysium which is a special connectivetissue which, among other things, contains the collagens I, III and V,elastic fibers, non-collagenic proteins such as fibronectin andproteoglycans, the serums may be tested for IgG and IgA antibodiesagainst gliadin using ELISA, and for IgG and IgA antibodies againstendomysium using indirect immunofluorescence. While antibodies againstgliadin are not sufficiently specific for sprue, high sensitivity andspecificity (97-100%) are reported for the IgA Ab against endomysium.However, esophagus sections from primates are required for theimmunofluorescence detection. At present, attempts are made to detectthe endomysium antibodies on umbilical cord material as well.

[0014] In 1984 Maury and Teppo (Lancet, 1984; 20.892-894) described a 90kDa mannose-rich glycoprotein (20% of sugar content) as a component ofthe normal skin and of the small intestine mucosa. They were able toidentify circulating IgG immunocomplexes able to recognize said protein,in 10 celiac patients out of 20 examined, and in 7 cases out of 12patients affected by herpes dermatitis (a disease considered to be anepidermal evidence of the celiac disease). The 90 kDa describedglycoprotein is characterized by having a 20% mannose content, whereastTG appears to be unglycosilated, notwithstanding the presence of 6glycosilation sites (Ichinose et al., J. Biol. Chem., 1990,265:13411-13414).

[0015] In 1986, Maury et al. (see Gut, 1986, 27:147-152) examined saidantibodies by an ELISA test with sera of celiac disease affectedpatients and control sera. Celiac disease affected patients showedantibody levels significantly higher (p<0.001) than those revealed incontrol sera. Said levels decrease to normal values further to agluten-free diet treatment. No correlation with anti-reticulinantibodies has been proved.

[0016] With a timely diagnosis and a strict observance of a gluten-freediet, the disease may be maintained in remission and thus, the increasedrisk of malignant tumors of the patients can also be lowered to a normalvalue. Therefore, there is great interest in developing a suitabledetection assay for sprue. Because the group of individuals bearing alatent sprue also belong to the high-risk group, all of the individualsin question (especially, the next of kin), and ultimately, all theschool children, as is presently taken into consideration in Italy,should be examined using a sensitive, specific, easily feasible, andlow-cost assay.

[0017] To date, however, large-scale screening programs failed as aresult of the following problems:

[0018] The invasive duodenal biopsies of symptom-free persons areunconscionable and exceedingly expensive.

[0019] An ELISA detection based on antibodies against gliadin isscarcely useful as a result of its poor specificity.

[0020] The immunofluorescence detection of IgA class endomysiumantibodies, which is based on primate esophagus, is too expensive as ageneral screening method. Furthermore, the assessment is subjective anddoes not permit identification of sprue patients having an IgAdeficiency (2% of the patients).

[0021] To date, therefore, a non-invasive, specific, quantitative,rapid, easily, and inexpensively feasible detection assay forsprue/coeliac disease and therapy control thereof does not exist.

[0022] This problem is solved by the present invention. Based on thesurprising finding that tissue transglutaminase (tTG, EC 2.3.2.13) isthe autoantigen of sprue, an immunological method according to claims 1through 6 for detecting antibodies against tTG and tTG-containingcompounds from body fluids, particularly from serum was developed, whichmethod not only permits diagnosing sprue or coeliac disease, but alldiseases accompanied by an immune reaction against tTG, tTG-containingcompounds, the antigenic structures, immunoreactive sequences oranalogues thereof.

[0023] The tissue transglutaminase belongs to the class oftransglutaminases. The TGs (EC 2.3.2.13) are enzymes catalyzing an acyltransfer depending on Ca²⁺, the γ-carboxamide groups of peptide-bondedglutamine residues acting as acyl donors. Primarily protein-bondedlysine residues function as acyl acceptors, as that the transfer resultsin an ε-(γ-glutamyl)lysine bond. The substrate specificity of the TGswith respect to the acyl donors is very high (depending on the aminoacid sequence), whereas an exceptionally wide Spectrum of acceptors isavailable (Folk J. E. Annu. Rev. Biochem., 1980; 49:517-531). Thecovalent peptide bonds formed are highly stable and protease-resistant,resulting in an increased resistance of the crosslinked proteins tochemical, enzymatic or physical effects.

[0024] Also, the widespread occurrence of various TGs in miscellaneousorgans, tissues, in plasma and interstitial body fluids correlates withthe occurrence of transglutaminase-modified proteins in blood clots, oncell membranes, in the horny layer of the epidermis, in hair, nails, andin the extracellular matrix (Greenberg C. S. et al FASEB J., 1991;5:3071-3077).

[0025] The described transglutaminases may be distinguished by theirphysical properties, their location in the body, and their primarystructure.

[0026] The tissue TG (tTG) is also referred to as cellular, erythrocyte,endothelial, cytoplasmatic, liver, or type II TG, and is a monomerhaving a molecular weight of 75-85 kDa.

[0027] The complete amino acid sequence comprising 687 residues wasderived from the cDNA. At the protein level, there is an 84% homologybetween the human enzyme and the enzyme from mouse macrophages and an81% homology between the human and guinea pig enzyme. Frequently,nucleotide exchanges between these species have no effect on the aminoacid sequence. The active center is highly conserved, with a markedprotein homology between the three species (49 out of 51 residues beingidentical), and a high degree of protein homology (75%) to the a-subnitof Factor XIII (see Gentile V. et al. J. Biol. Chem., 1991; 266:478-483;Greenber C. S. et al. FASEB J., 1991; 5:3071-3077).

[0028] Neither is there a signal peptide nor glycosylation, andapparently, despite multiple cysteine residues, there are no disulfidebridges. Using fluorescence hybridizations, the gene for human tissuetransglutaminase was localized on chromosome 20q12 (Gentile V. et al.,Genomics, 1994; 20:295-297). Although the mechanism of enzyme liberationstill is not clear, there is unequivocal evidence that tTG with itsintracellular ubiquity attains important functions within theextracellular matrix (ECM). Moreover, the Ca²⁻ intracellularconcentration required for tTG activity is not likely to be reachedunder physiological conditions, whereas sufficiently high Ca²⁻concentrations are present in the extracellular range (Gentile V. etal., J. Cell Biol., 1992; 119:463-474).

[0029] Several investigations establish an association of tTG with thefibronectin ECM protein. In addition to fibronectin, the ECM moleculesnidogen, the N-terminal procollagen III peptide, the collagens V and XI,osteonectin, which is a microfibril-associated glycoprotein, highmolecular weight dermatan sulfate proteoglycan, and the galectin 3lectin could be identified as specific substrates for tTG.

[0030] Indications for an important role of tTG in wound healing werealso obtained from immunofluorescence studies on cultivated WI38 cells(lung embryonal fibroblasts) which do not exhibit extracellular tTGactivity under normal conditions but effect extracellular deposition ofthe enzyme upon artificial wound generation. The possibly passiverelease of the enzyme from damaged cells is followed by an initiallynon-covalent binding to the ECM, particularly to fibronectin andfibrillary collagens, where the enzyme is catalytically active for somehours (Upchurch, H. F. et al.; J. Cell. Physiol., 1991; 149:375-382).Using a rat model, likewise after artificial wound generation, a 5 dayincreased tTG activity was detected (Bowness, J. M. et al. Biochem.Biophys. Acta; 1988; 967:234-240). Also, when incubating humanerythrocyte lysates with plasma, a strong affinity of the liberated tTGto fibronectin could be demonstrated (Loraud, L. et al. Proc. Natl.Accad. Sci USA 1988; 85 : 1057- 1059). All the findings indicate thatthe tTG bound to ECM assumes a central role in the early phase of woundhealing and, in particular, contributes to fibrin stabilization togetherwith Factor XIII, forming a protective layer and a stable adhesivesubstrate around the damaged cells by crosslinking of extracellularproteins. To date, no enzymes capable of cleaving the tTG-catalyzed,enormously stable crosslinkages to the proteins could be detected invertebrates.

[0031] Based on the finding that tTG is the autoantigen of sprue, theinvention is also directed to the use of tTG, tTG-containing compounds,the antigenic structures, immunoreactive sequences or analogues thereofin the diagnosis and therapy control of diseases accompanied by animmune reaction against said compounds. In particular, acuteinflammatory diseases such as pneumonia, glomerulonephritis, virushepatitis, or chronically inflammatory diseases, such as Morbus Crohn,Colitis ulcerosa, or autoimmune diseases such as autoimmune hepatitis,Sjoegren syndrome, Wegener's granulomatosis, rheumatoid arthritis,idiopathic organ fibrosis, such as lung fibrosis can be diagnosed inthis way. Especially suitable is the detection assay for the diagnosisand therapy control of sprue. Since this assay can be performed quicklyand cost-favorably, it permits efficient screening of the population fortTG antibodies.

[0032] The tTG used according to the invention may be of animal,synthetic or recombinant origin, and the same applies for thetTG-containing compounds which, in addition, may also involve a combinedorigin (e.g., animal tTG in association with a synthetic peptide). Inthe meaning of the invention, tTG-containing compounds are understood tobe chemical compounds of tTG with proteins, or analogues thereof. In themeaning of the invention, tTG analogues or analogues of thesetTG-containing compounds are understood to be all those antigenicstructures that undergo an immune reaction with antibodies against tTGor tTG-containing compounds, e.g., synthetic peptides. Immunoreactivesequences are understood to be fragments of tTG or tTG-containingcompounds produced by proteolysis, synthesis or genetic engineering, aswell as variants obtained by amino acid exchange.

[0033] The immunological detection according to the invention isperformed using well-known methods. Thus, any direct (e.g., using asensor chip) or indirect procedure may be used in the detection ofpatient antibodies.

[0034] In the direct procedures, binding of the antibodies to bedetected to the antigen is determined through the alteration of chemicalor physical properties, so that subsequent detection steps usinglabelled binding species are not required.

[0035] According to the invention, it is preferred to detect the tTGantibodies using an immunoassay, preferably a solid phase immunoassay,with direct or indirect coupling of a reactant to an easily detectablelabelling substance. More preferably, detection may be carried out usingan ELISA, an RIA, or an immunofluorescence assay. The proceduresinvolved in such detection methods are well-known to a person skilled inthe art.

[0036] In an ELISA, for example, the antigen, in the present case, e.g.,tTG is bonded directly or indirectly to a carrier substance such aspolystyrene. Following incubation with the antibodies to be detected,e.g., from serum of patients, the antigen-bonded antibodies are detecteddirectly or indirectly using enzyme-coupled substances. These substancesmay be antibodies, fragments of antibodies, or high-affinity ligands,such as avidin which binds to a biotin label. For example, peroxidase,alkaline phosphatase, β-galactosidase, urease, or glucose oxidase arepossible as enzymes. The bonded enzymes and thus, the bonded tTGantibodies, for example, may be quantified by adding a chromogenicsubstrate.

[0037] In a radioimmunoassay, the antigen, e.g., tTG is likewise bondeddirectly or indirectly to a carrier substance such as polystyrene.Following incubation with the antibodies to be detected, e.g., fromserum of patients, the antigen-bonded antibodies are detected usingsubstances bearing a radioactive label, e.g., ¹²⁵I. These substances maybe antibodies, fragments of antibodies, or high-affinity ligands, suchas avidin which binds to a biotin label. The bonded radioactivity may bequantified using a suitable measuring instrument.

[0038] In an immunofluorescence assay, the antigen-bonded antibodies aredetected according to the same principle, using substances which carry afluorescent label, e.g., fluorescein isothiocyanate (FITC). Thesesubstances may be antibodies, fragments of antibodies, or high-affinityligands, such as avidin which binds to a biotin label. The bonded amountof fluorescent dye is then quantified using a suitable measuringinstrument.

[0039] According to the invention, it is also possible to detect thepatient antibodies in an agglutination assay or a gel diffusion assay.These detection assays are also familiar to a person skilled in the art.Thus, in a gel diffusion assay, the antigen or antibody solutions, forexample, are placed into closely adjoining wells of agar or agaroseplates. In the present case, the antigen solution may be, e.g., the tTGsolution, and the antibody solution may be blood serum, for example. Asthe substances diffuse out of their wells, concentration gradients areproduced starting from the wells. If the overlapping antigen andantibody concentrations in the gel fall within specific ratios, and theantibody solution contains antibodies against the antigen, perceptibleprecipitates are formed in the gel.

[0040] In the agglutination assay, antigen(e.g., tTG)-bearing particles,e.g., from latex or polystyrene, are crosslinked by antibodies, e.g.,from serum. The aggregates produced may be detected using turbidimetry,for example.

[0041] According to the invention, it is particularly preferred toperform the detection in the serum of sprue patients using anIgA-specific or IgG-specific ELISA. It was found that the tTG-based,newly developed ELISA detection of IgA antibodies in the serum of spruepatients is excellently suited for the diagnosis and therapy control ofsprue as a result of its high sensitivity and specificity. This is alsoapparent in the follow-up of the treated patients (drop in titer duringtherapy). A comparison of the ELISA data of the invention with theimmunofluorescence evaluations of third persons (detection of IgAanti-endomysium) shows good conformity. Incongruities especially occurwith low antibody titers which, however, are a result of the indirectimmunofluorescence being deemed as top level standard up to now. Interalia, this is due to the subjective evaluation and the non-specificconcurrent reactions of this prior art method.

[0042] The corresponding detection based on antibodies from otherclasses, exemplified by the IgG antibodies, is suitable for identifyingsprue patients with IgA deficiency and for the examination of otherdiseases accompanied by an immune reaction against tTG.

[0043] Another improvement of this direction method results when usingpurified tTG from guinea pigs, human tTG, sequences or analoguesobtained by proteolysis or genetic engineering, as well as syntheticimmunogenic tTG peptides in the test system. An ELISA for the diagnosisand follow-up of other diseases accompanied by an immune reactionagainst tTG will be described in Example 3.3.

[0044] The invention is also directed to an oral pharmaceutical agentaccording to claim 10 for the treatment of diseases accompanied by animmune reaction against tTG, tTG-containing compounds, the antigenicstructures, immuno-reactive sequences or analogues thereof. Preferably,the oral administration form is a tablet or a capsule where an oraltolerance is produced by administering tTG, tTG-containing compounds,the antigenic structures, immunoreactive sequences or analogues thereof.On the one hand, said oral tolerance is achieved by oral supply of theautoantigen, and on the other hand, there is a so-called “bystandereffect”: if the autoantigen inducing the disease in unknown, anotherantigen contacting the immune system in the target organ can be used inoral therapy in some cases. This antigen then is capable of locallystimulating the antigen-specific suppressor T cells, thereby suppressinga systemic immune response. Only at higher antigen doses, an anergy ofautoreactive T cells is induced.

[0045] Oral tolerance is the practical method of treating miscellaneousautoimmune diseases.

[0046] The pharmaceutical agent of the invention is preferably used inthe treatment of sprue, but also for other chronic inflammatoryintestinal diseases and autoimmune hepatitis.

[0047] According to the invention, tTG, tTG-containing compounds, theantigenic structures, immunoreactive sequences or analogues thereof areadministered at a dosage of 0.01-100 mg/kg body weight.

[0048] The pharmaceutical agent of the invention may optionally containpharmaceutically tolerable adjuvants, such as fillers, lubricants,disintegrants, binders, or release agents normally used in galenism. Theratio of the pharmaceutically adjuvants may vary within wide limitsdepending on the selected content of active ingredient, and is from 0.1to 20% by weight in each case.

[0049] In particular, the advantages achieved with the present inventioncan be seen in a detection assay for sprue and the therapy controlthereof, which assay is non-invasive, highly specific, and directedimmediately against the agent associated with the disease. Moreover, amajor advantage of the assay that has been developed is the rapid, easyand cost-favorable practicability, as well as the possibility ofstandardizing between different laboratories. Thereby, the assay permitsefficient screening of the population for antibodies directed againsttTG.

[0050] Also, as a result of the objectiveness of the assay data, thepotential of quantitative evaluation is superior compared to theimmunofluorescence evaluation involving subjective features. Inaddition, immunofluorescence evaluations, particularly with low titers,are hampered by non-specific concurrent reactions. By using the specificauto-antigen in the test system, the non-specific reactions on esophagusmaterial of primates or on umbilical cords in immunofluorescence can beeliminated to the furthest possible extent.

[0051] Since the assay is applicable to IgA class antibodies as well asother classes of antibodies, sprue patients with IgA deficiency are alsoidentified. The detection based on antibodies against tTG is alsosuitable for the identification, examination and therapy control ofother diseases accompanied by an immune reaction against tTG.

[0052] Also, as a result of the identification of tTG as an autoantigenof sprue, it is possible to use same in its entity or the immunoreactiveepitopes thereof (sequences, analogues or synthetic peptides produced byproteolysis or genetic engineering) in the oral therapy of sprue andother diseases accompanied by an immune response to tTG.

EMBODIMENTS EXAMPLE 1

[0053] Isolation and Characterization of the Autoantigen

[0054] 1.1. Immunofluorescence, APAAP Stainings

[0055] The stainings were carried on various cell lines fixed in 100%methanol for 2 min at −20° C.

[0056] In the immunofluorescence detection, the preparations wereincubated with sprue and control serums, respectively, washed anddetected using a TRITC-labelled anti-human IgA from rabbit (Schuppan etal., J. Biol. Chem. 1990; 265, 8823-32).

[0057] APAAP labelling was carried out after incubating the cells withthe sprue serums, washing and subsequent detection using the APAAPcomplex (Cordell J. L. et al., J. Histochem. Cytochem. 1984; 32,219-229).

[0058] Here, HT1080 (human fibrosarcoma cells) WI38 (human, lungembryonal fibroblasts), Hep 1 and HepG2 (hepatocarcinoma cells) showedunequivocally positive cytoplasmatic signals with the patient serums,whereas normal serums or pretreatment with human IgA did not show anylabelling. Human prepuce fibroblasts, human rhabdomyo-sarcoma (RD)/ratIto/rat Morris hepatoma, and dog MDCK cells only showed from very faintto negative reactions.

[0059] 1.2 Metabolic Cell Labelling and Immunoprecipitation of theAutoantigen

[0060] Characterization and isolation of the autoantigen were carriedusing HT1080 cells.

[0061] The cells were cultivated with L-alanyl-L-glutamine, 10% fetalcalf serum (FCS, Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin(Seromed) in Dulbecco's modified Eagle medium (DMEM, Gibco) at 37° C.and 8% CO₂. For metabolic labelling, the cells were transferred intoculture dishes of 5 cm in diameter, and once a −90% confluence wasreached, they were kept in a medium free of methionine and FCS,whereafter this medium was replaced by 3 ml of an FCS-free mediumcontaining ³⁵S-methionine (0.2 mCi, Expre³⁵S³⁵S, NEN-Dupont). After16-20 hours of incubation, the supernatant was removed. The cells werewashed with phosphate buffer (PBS, Seromed), and subsequently lysed in 3ml of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 0.5%IGEPAL CA-630 non-ionic detergent [Sigma], Complete® protease inhibitor[Boehringer], pH 7.5). Thereafter, an immunoprecipitation usingCNBr-activated Sepharose 4B (Pharmacia) was carried out both with themedium and the cell lysate.

[0062] Activation and binding to Sepharose were conducted according tothe manufacturer's instructions. After swelling and washing in 1 mM HCl,pH 2.5, the CNBr-activated Sepharose was incubated with an antibody fromrabbit directed against human IgA (Dianova, 2.4 mg antibody/ml ofSepharose) in 0.1 M NaHCO₃, 0.5 M NaCl, pH 8.3, at 4° C. overnight.Non-bonded antibodies were removed by washing with the coupling buffer,and unoccupied binding sites were saturated by adding 1 M ethanolamine,pH 9.0, at room temperature, 2 hours. Thereafter, the Sepharose waswashed 3 times alternately (10×Vol. each) with 0.1 M sodium acetate, 0.5M NaCl, pH 4.0, and 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0, followed byincubation of the Sepharose with serums from sprue patients and healthypersons, respectively (0.5 ml serum/ml Sepharose), in coupling buffer(50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH 8.0) at 4° C.overnight. Excess serum antibodies were removed by 3 washings withcoupling buffer.

[0063] Each time, 1 ml of HT1080 medium or cell lysate (about 5×10⁴cells) of the metabolic-labelled cells was pre-incubated with 50 μL ofC14B Sepharose (Pharmacia) for 30 min at room temperature, to removenon-specifically bonding proteins. Following centrifugation (10,000×g, 5min, 4° C.), each of the supernatants were incubated with agitation at4° C. overnight with 50 μl of the Sepharose to which IgA from thepatients and control persons, respectively, had previously been bonded.The Sepharose pellets were then washed each time with 3×1 ml washingbuffer (10 mM Tris-HCl, 1% IGEPAL CA-630 [Sigma], 0.5% sodiumdeoxycholate, 0.1% sodium lauryl sulfate, Complete® [Boehringer], pH8.0), followed by 1 ml 10 mM Tris-HCl, pH 8.0. Then, the pellets weretaken up in SDS assay buffer, incubated for 5 min at 95° C. underreducing or non-reducing conditions, separated on a 10-12.5% SDSpolyacrylamide gel (Lämmli, U.K., Nature 1970; 227, 680-685), anddetected using autoradiography (FIG. 1).

[0064] In further examinations, the bonded high molecular weight proteinfrom the medium was found to be fibronectin which, inter alia, isnon-specifically bonded to Sepharose.

[0065] However, a cell-associated protein of 85 kDa could beprecipitated with all the 30 sprue serums used so far in this way,whereas the same was not possible with 15 control serums, includingnormal serums, serums from patients with Colitis ulcerosa and Sjoegrensyndrome. It was concluded from this fact that this protein representsthe essential autoantigen of sprue.

[0066] By means of protein staining of the gels using silver nitrate(Henkeshoven, J. et al., Electrophoresis 1985; 6, 103-112), a sharpprotein band was assigned to the autoradiographically visible 85 kDaband.

[0067] 1.3. Isolation and Purification of the 85 kDa Autoantigen

[0068] In order to isolate larger quantities of the autoantigen, a totalof 65 culture dishes (175 cm² each) with HT1080 cells (about 10⁹ cells)were cultivated. A short time before reaching confluence, the medium wasreplaced by an FCS-free medium, followed by incubation for another 16-20hours in a CO₂ incubator. Lysis and immunoprecipitation, respectively,were effected as described above. The Sepharose pellet was incubated ina total of 4.5 ml SDS assay buffer with 2% DL-dithiothreitol (Sigma) for5 min at 95° C. to detach bonded proteins and subsequently scrutinizedin the analytic SDS polyacrylamide gel.

[0069] For further purification of the autoantigen, theimmunoprecipitate was separated via elution electrophoresis as follows,using a Prep Cell (Model 491 BIO-RAD): 4.5 ml of the protein mixture wasplaced on top of a round gel (outer diameter: 3 cm) consisting of 6.5 cmseparation gel (8% polyacrylamide, pH 8.8) and 1.5 cm collecting gel (4%polyacrylamide, pH 6.8) and separated by electrophoresis. The individualproteins were collected in the elution buffer (25 mM Tris-HCl, 0.1 Mglycine, 0.01% SDS, pH 8.3) as fractions of 1.2 ml each (0.8 ml/min).The eluted fractions were scrutinized in the SDS PAGE, and the fractionscontaining the desired protein (about 15 ml) were combined andconcentrated to about 1 ml overall volume using ultrafiltration (AmiconCentriprep-50, at 1,000×g).

[0070] 1.4. Protease Digestion of the Autoantigen

[0071] Amongst several tested proteases, endoproteinase Asp-N(sequencing grade, Boehringer Mannheim) was determined to be suitablefor fragmentation, because it permitted a largely reproducible cleavagepattern with relatively well-separable fragments. The enzyme/substrateconcentration was adjusted to 1:100, and the digestion was carried outfor 30 min at 37° C.

[0072] 1.5. Transfer to PDVF Membrane

[0073] Following digestion of the purified autoantigen, the peptidefragments were separated on a preparative 10% Tricine gel (Schägger, H.et al., Anal Biochem. 1987; 166, 368-379) (FIG. 2) and transferred at 4°C. onto a PVDF membrane (polyvinylidene difluoride, Immobilon™,Millipore) in a semi-dry fastblot procedure using graphite-containingelectrode plates (Fastblot 332/33, Biometra). To this end, the followinglayers were placed on the anode plate: 1.) a filter paper soaked inanode buffer 1 (300 mM Tris-HCl, 20% methanol, pH 10.4), 2.) a filterpaper soaked in anode buffer 2 (30 mM Tris-HCl, 20% methanol, pH 10.4),3.) the PVDF membrane activated in methanol and pre-equilibrated inanode buffer 2, 4.) the Tricine gel, 5.) two filter papers soaked incathode buffer (25 mM Tris-HCl, 40 mM ε-amino-n-caproic acid, 20%methanol, pH 9.4), 6.) the cathode plate. The transfer was carried outover 35 min at 180 mA.

[0074] Thereafter, the PVDF membrane was stained in 0.1% Coomassie BlueServa R-250, 50% methanol for 5 min, bleached with 50% methanol, 10%acetic acid, washed thoroughly with distilled water, and air-dried. Thecharacteristic bands of the digested autoantigen at 10 kDa, 14 kDa, 16kDa, and 25 kDa were cut out carefully and subjected to a firstsequencing at the N terminus.

[0075] 1.6 Edman Degradation

[0076] (According to Edman and Henschen in: Needleman, S. B.: ProteinSequence Determination, Springer Verlag, Berlin. 1975; 232-279)

[0077] Sequencing using an Applied Biosystems 4778-Sequenator resultedin three amino acid sequences which were compared with the Swiss Prot 31data base (by PC/GENES, IntelliGenetics). From these, an unequivocalassignment of the three fragments to human tissue transglutaminase (tTG,EC 2.3.2.13, protein glutamine γ-glutamyl-transferase) could be madewith minimum discordance. The indications are given using the “oneletter code”; X represents no identification:

[0078] t-Transglutaminase: 28′REKLVVRRGQPFW

[0079] 10 kDa fragment: REKLVVRRGQPF(S)

[0080] t-Transglutaminase: 581′DLYLENPEIKIRILG

[0081] 14 kDa fragment: DLYLENPEIXIXILG

[0082] t-Transglutaminase: 438′DITHTYKYPE

[0083] 16 kDa fragment: DITLTYQYP(V)

[0084] No equivocal sequence could be assigned to the 25 kDa fragment,because it was a peptide mixture.

EXAMPLE 2

[0085] Confirmation of Tissue Transglutaminase (tTG) as SprueAutoantigen

[0086] 2.1. Immunoprecipitation of Guinea Pig tTG

[0087] Being commercially available and having sequence homology (<80%)to human tTG, the tTG from guinea pig liver (Sigma) was first separatedby gel electrophoresis, in order to check the purity thereof. Inaddition to several other proteins, tTG, being about 50%, represents oneof the major bands.

[0088] Although human tTG having 687 amino acids differs only slightlyfrom the guinea pig protein having 690 amino acids, these two proteinsshow highly different migrating behavior in the SDS polyacrylamide gel.While the protein of animal origin appears at 75-80 kDa as expected, thehuman protein shows a distinctly less rapid migration, pretending anapparent molecular weight of 85 kDa as described in literature Gentile,V., et al., J. Biol. Chem. 1991; 266, 478-483), despite apparentlylacking N-glycosylation.

[0089] The reactivity of the human autoantibody from Sprue serums withguinea pig tTG was tested in an immunoprecipitation. To this end, 4 μgof tTG (Sigma) in 500 μl of lysis buffer and 0.5% bovine serum albuminwere agitated at 4° C. overnight with sprue IgA coupled to 4B Sepharose,washed, boiled in SDS assay buffer under reducing conditions, andseparated on a 10% polyacrylamide gel (cf. 4.1.2.). Here, specificprecipitation of the expected band (m.w. 80 kDa) occurred, but none ofthe impurity.

[0090] 2.2 Confirmation of tTG as Autoantigen in a Western Blot

[0091] Following separation of 2 μg of guinea pig tTG on an SDS gel andtransfer onto nitrocellulose, the blot was blocked in PBS, 2% low-fatskim milk powder, 0.3% Tween 20, pH 7.3, at 4° C. overnight. This wasfollowed by a one hour incubation with sprue serum ({fraction (1/200)})in the same buffer, three washing steps and a one hour incubation withalkaline phosphatase-coupled rabbit antibodies against human IgA({fraction (1/500)}). The blots were washed in PBS and developed withNitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate assubstrate (Blake, M. S., et al., Anal. Biochem. 1984; 136, 175-179).

[0092] The 75-80 kDa band gave an unequivocal positive signal with thesprue serum, which is another proof that the serums of sprue patientscontain IgA class antibodies against tTG, whereas control serums did notgive any signal.

[0093] 2.3. Confirmation of tTG as Endomysium Autoantigen Using IndirectImmunofluorescence

[0094] Esophagus tissue sections from primates (Euroimmun, Germany) wereused in the indirect detection of the IgA antibodies against endomysiumin sprue serums and their inhibition by tTG. Following pre-incubation of10 μl of patient serum diluted {fraction (1/320)} in PBS with 0.5 or 10μg of tTG from guinea pigs (Sigma) and 10 μg of BSA (Sigma) for 1 hourat room temperature, incubation thereof with said esophagus sections wasperformed for 1 hour at room temperature in humid atmosphere. Sprueserum ({fraction (1/320)}) and serums of healthy individuals ({fraction(1/50)}) were used as positive and negative controls, respectively.After the sections had been washed three times in PBS/0.2% BSA andair-dried, the detection of the autoantigen with a TRITC-labelled rabbitantibody against human IgA (Dianova), diluted {fraction (1/50)} in PBS,was effected for 1 hour at room temperature. Excess antibodies wereremoved by successive washings with PBS/0.2% BSA, PBS, and distilledwater.

[0095] The patient serum showed clear staining of the ECM by the IgAclass antibodies which were inhibited by adding increasingconcentrations of tTG, but not by pre-incubation with BSA. The controlusing serum of healthy individuals did not show any staining of theesophagus sections.

EXAMPLE 3

[0096] 3.1. Development of a Sprue-Specific ELISA with IgA Antibodiesfor the Diagnosis and Follow-up of Sprue

[0097] 1 μg of guinea pig transglutaminase (Sigma T-5398) in 100 μl PBSwas pipetted into each well of polystyrene microplates (GreinerLabortechnik, 96 Wells) and incubated for 2 h at 37° C. under slightlyrotating motion. Non-bonded tTG was removed by washing with PBS (3×200μl), the free bonding sites of the wells were blocked with 1% bovineserum albumin (Sigma) in 250 μl of PBS at 4° C. overnight. After washingwith PBS/0.1% Tween 20 (3×200 μl), the wells were incubated withsequential serum dilutions in PBS/0.1% Tween 20 (100 μl) for 1 hour atroom temperature under slightly rotating motion, washed with PBS/0.1%Tween 20 (3×200 μl) and subsequently incubated with aperoxidase-conjugated rabbit antibody directed against human IgA(Dianova) ({fraction (1/400)} in 100 μl PBS/0.1% Tween 20) for 1 hour atroom temperature. After washing with PBS (3 times), a 30 min incubationat room temperature in the dark, using 200 μl 0.1 M citrate buffer, 17.6mM H₂O₂, 5.5 mM o-phenylenediamine hydrochloride (Sigma), pH 4.2, andsubsequent detection of the formed dye in an ELISA reader (MRX, DynatechLaboratories) at 450 nm were performed.

[0098] 20 serums of sprue patients were tested before and after therapyusing a gluten-free diet, i.e., in the active and less active phases ofthe disease. The test system was found to be highly sensitive, with agood correlation of the values to the sprue active phase. Thetherapeutical success as a result of observing a diet was reflected in adecrease of the IgA antibodies against tTG. The high specificity becameobvious in the low extinction (background level) of the control serumsof healthy individuals, patients with Colitis ulcerosa, liver cirrhosis,miscellaneous tumors, Sjoegren syndrome, etc. (FIG. 3).

[0099] 3.2. Development of an ELISA Using Antibodies from Other Classesfor the Diagnosis and Follow-up of Sprue, with IgG Antibodies as Example

[0100] As about 2% of the sprue patients have an IgA deficiency, theserums were tested for their sensitivity and specificity of IgGantibodies against tTG. The ELISA was performed as in 3.1., only theperoxidase-coupled anti-human IgA antibody (Dianova) was replaced by ananti-human IgG antibody (Dianova). With respect to their sensitivity,the values of the sprue patients both before and after gluten-free dietwere corresponding to the data obtained with IgA antibodies.

[0101] Some of the control serums showed slightly increased values,which corresponds to earlier findings of a reduced specificity of theendomysium antibodies in the in direct immunofluorescence of the IgGclass (FIG. 4).

[0102] 3.3. Development of an ELISA for the Diagnosis and Follow-up ofOther Diseases Accompanied by an Immune Reaction against tTG, with IgGAntibodies as Example

[0103] The ELISA was performed as described in 3.2.

[0104] The serums of patients with chronically inflammatory orautoimmune diseases (Colitis ulcerosa (C.U.), Morbus Crohn, acuteautoimmune hepatitis) showed from slightly to moderately increasedvalues.

[0105] Thus, by using the IgG-specific ELISA for autoantibodies againsttTG, the diagnosis and therapy control of patients suffering fromdiseases accompanied by an immune reaction against tTG is possible.

EXAMPLE 4

[0106] A New Function of Tissue Transglutaminase (tTG) in theCrosslinking of Gliadin

[0107] While a wide spectrum of acyl acceptors is available for thetTG-catalyzed reaction, only few molecules are capable of acting as acyldonors. In an in vitro experiment, the tTG-mediated incorporation ofradioactively labelled putrescine into gliadin and thus, the function ofgliadin as donor substrate of tTG could be established. In 160 μl ofbuffer (0.1 M Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, pH 7.5), 1 μg ofsubstrate (gliadin or control proteins such as albumin), 2 μCi[³H]-putrescine, and 1 μg of tTG (from guinea pigs, Sigma) wereincubated for 2 hours at 37° C. The reaction was stopped by adding 100μl of 50% trichloroacetic acid (TCA), and the proteins were precipitatedat 4° C. overnight. Following centrifugation, the pellets were washedwith 10% TCA, dissolved in SDS assay buffer and, on the one hand,separated in an SDS PAGE and, on the other hand, used in a scintillationcount. While no incorporation of putrescine could be determined with thecontrols, gliadin shows clear incorporation of [³H]-putrescine both inthe scintillation count data and in the SDS PAGE, proving that gliadinis an excellent substrate for tTG.

Abbreviations

[0108] Ab: Antibody

[0109] APAAP: Alkaline phosphatase anti-alkaline phosphatase

[0110] BSA: Bovine serum albumin

[0111] cm: Centimeter

[0112] DMEM: Dulbecco's modified Eagle Medium

[0113] EC: Enzyme Commission

[0114] ELISA: Enzyme-linked immunosorbent assay

[0115] ECM: Extracellular matrix

[0116] FCS: fetal calf serum

[0117] h: hour(s)

[0118] H₂O₂: Hydrogen peroxide

[0119] HLA: Human lymphocyte antigens

[0120] IEL: Intraepithelial lymphocytes

[0121] Ig: Immunoglobulin

[0122] kDa: Kilodalton

[0123] M: Molar

[0124] mA: Milliampere

[0125] MHC: Major histocompatibility complex

[0126] min: Minute(s)

[0127] mM: Millimolar

[0128] M_(r): Relative molecular mass

[0129] μg: Microgram

[0130] μl: Microliter

[0131] PAGE: Polyacrylamide gel electrophoresis

[0132] PBS: Phosphate buffer

[0133] PLA₂: Phospholipase A₂

[0134] PVDF: Polyvinylidene difluoride

[0135] SDS: Sodium dodecyl sulfate

[0136] TCA: Trichloroacetic acid

[0137] TGF: Transforming growth factor

[0138] Tris: Tris(hydroxymethyl)aminomethane

[0139] tTG: Tissue transglutaminase

1. A method for immunologically identifying the celiac disease,characterized in that anti-tTG antibodies in body fluids are detected byan immunoreation with a tissue transglutaminase (tTG), withtTG-containing compounds, with antigenic structures thereof, withimmunoreactive sequences or analogs thereof.
 2. Method according toclaim 1, characterized in that human IgA and/or IgG antibodies aredetected.
 3. Method according to claim 1 or 2 characterized in that thetTG or tTG-containing compounds are of human, animal, synthetic orrecombinant origin.
 4. The method according to one of claims 1 through3, characterized in that the detection is performed using a per se knownimmunoassay, preferably with direct or indirect coupling of one reactantto a well-detectable labelling substance.
 5. The method according toclaim 4, characterized in that the detection is carried out on a solidphase.
 6. The method according to claim 4, characterized in that thedetection is carried out using an ELISA, RIA or an immunofluorescenceassay.
 7. Use of tTG, tTG-containing compounds, the antigenicstructures, immunoreactive sequences or analogues thereof in thediagnosis or therapy control of auto-immune diseases.
 8. The useaccording to one of claim 7 for the diagnosis or therapy control ofcoeliac disease.
 9. An oral pharmaceutical agent including tTG,tTG-containing compounds, the antigenic structures, immunoreactivesequences or analogues thereof and optionally, pharmaceuticallytolerable adjuvants, for the treatment of coeliac disease.
 10. Use oftTG-containing compounds, the antigenic structures, immunoreactivesequences or analogues thereof in the production of oral pharmaceuticalagents for the treatment of coeliac disease.